Checkpoint inhibitors have demonstrated efficacy in patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, the majority of patients do not benefit from these agents. To improve the efficacy of checkpoint inhibitors, intratumoral (i.t.) injection with innate immune activators, TLR7 and TLR9 agonists, were tested along with programmed death-1 receptor (PD-1) blockade. The combination therapy suppressed tumor growth at the primary injected and distant sites in human papillomavirus–negative (HPV-negative) SCC7 and MOC1, and HPV-positive MEER syngeneic mouse models. Abscopal effects and suppression of secondary challenged tumor suggest that local treatment with TLR agonists in combination with anti–PD-1 provided systemic adaptive immunity. I.t. treatment with a TLR7 agonist increased the ratio of M1 to M2 tumor-associated macrophages (TAMs) and promoted the infiltration of tumor-specific IFNγ-producing CD8 ⁺ T cells. Anti–PD-1 treatment increased T cell receptor (TCR) clonality of CD8 ⁺ T cells in tumors and spleens of treated mice. Collectively, these experiments demonstrate that combination therapy with i.t. delivery of TLR agonists and PD-1 blockade activates TAMs and induces tumor-specific adaptive immune responses, leading to suppression of primary tumor growth and prevention of metastasis in HNSCC models.

High-throughput sequencing of T cell receptor (TCR) genes is a powerful tool for analyses of antigen specificity, clonality and diversity of T lymphocytes. Here, we developed a new TCR repertoire analysis method using 454 DNA sequencing technology in combination with an adaptor-ligation mediated polymerase chain reaction (PCR). This method allows the amplification of all TCR genes without PCR bias. To compare gene usage, diversity and similarity of expressed TCR repertoires among individuals, we conducted next-generation sequencing (NGS) of TRA and TRB genes in peripheral blood mononuclear cells from 20 healthy human individuals. From a total of 267,037 sequence reads from 20 individuals, 149,216 unique sequence reads were identified. Preferential usage of several V and J genes were observed while some recombinations of TRAV with TRAJ appeared to be restricted. The extent of TCR diversity was not significantly different between TRA and TRB, while TRA repertoires were more similar between individuals than TRB repertoires were. The interindividual similarity of TRA depended largely on the frequent presence of shared TCRs among two or more individuals. A publicly available TRA had a near-germline TCR with a shorter CDR3. Notably, shared TRA sequences, especially those shared among a large number of individuals’, often contained TCRα related with invariant TCRα derived from invariant natural killer T cells and mucosal-associated invariant T cells. These results suggest that retrieval of shared TCRs by NGS would be useful for the identification of potential new invariant TCRα chains. This NGS method will enable the comprehensive quantitative analysis of TCR repertoires at a clonal level.

(1) Background: Comparable prognoses after definitive chemoradiation therapy (CRT) to surgery alone for esophageal squamous cell carcinoma (ESCC) have been previously reported; however, no robust prognostic markers have been established. The clonality of tumor-infiltrating lymphocytes (TILs) and tumor microenvironments (TMEs) in ESCC relapsed after CRT were examined to explore prognostic markers. (2) Methods: Clonality of TIL and TME were examined in ESCC with and without preceding CRT, as well as oral squamous cell carcinoma (OSCC) and healthy volunteers as controls. The clonality of TIL was assessed by T-cell receptor (TCR) α and β repertoire analyses and evaluated by diversity indices. The TME was assessed by quantitative polymerase chain reaction evaluating PD-L1 and CD8B. (3) Results: The clonal expansion of TIL was significantly induced within ESCCs and OSCCs, when compared to healthy volunteers, and was mostly induced within ESCCs after definitive CRT. Diversity indices of TIL were not associated with the prognosis, but the ratio of PD-L1 mRNA to CD8B mRNA in TME was significantly associated with a poor prognosis after salvage surgery (p = 0.007). (4) Conclusions: The clonal expansion of TIL is induced after definitive CRT for ESCC, and the ratio of PD-L1 mRNA to CD8B mRNA within tumor tissues is a prognostic marker candidate for salvage esophagectomy after CRT.

T-cell recognition of antigen epitopes is a crucial step for the induction of adaptive immune responses, and the identification of such T-cell epitopes is, therefore, important for understanding diverse immune responses and controlling T-cell immunity. A number of bioinformatic tools exist that predict T-cell epitopes; however, many of these methods highly rely on evaluating conventional peptide presentation by major histocompatibility complex (MHC) molecules, but they ignore epitope sequences recognized by T-cell receptor (TCR). Immunogenic determinant idiotopes are present on the variable regions of immunoglobulin molecules expressed on and secreted by B-cells. In idiotope-driven T-cell/B-cell collaboration, B-cells present the idiotopes on MHC molecules for recognition by idiotope-specific T-cells. According to the idiotype network theory formulated by Niels Jerne, such idiotopes found on anti-idiotypic antibodies exhibit molecular mimicry of antigens. Here, by combining these concepts and defining the patterns of TCR-recognized epitope motifs (TREMs), we developed a T-cell epitope prediction method that identifies T-cell epitopes derived from antigen proteins by analyzing B-cell receptor (BCR) sequences. This method allowed us to identify T-cell epitopes that contain the same TREM patterns between BCR and viral antigen sequences in two different infectious diseases caused by dengue virus and SARS-CoV-2 infection. The identified epitopes were among the T-cell epitopes detected in previous studies, and T-cell stimulatory immunogenicity was confirmed. Thus, our data support this method as a powerful tool for the discovery of T-cell epitopes from BCR sequences.

The element chromium (Cr) is a component of several types of alloys found in the environment, or utilized in dentistry, that may cause intraoral metal contact allergy. However, the pathological mechanism of intraoral Cr allergy remains unclear because there is no established animal model of Cr allergy in the oral mucosa. In this study, we established a novel murine model of Cr-induced intraoral metal contact allergy and elucidated the immune response in terms of cytokine profiles and T-cell receptor repertoire. Two sensitizations with Cr plus lipopolysaccharide solution into the postauricular skin were followed by a single Cr challenge of the oral mucosa to generate the intraoral metal contact allergy model. Histological examination revealed that CD3+ T-cells had infiltrated the allergic oral mucosa one day after exposure to the allergen. The increase in T-cell markers and cytokines in allergic oral mucosa was also confirmed via quantitative PCR analysis. We detected Cr-specific T-cells bearing TRAV12D-1-TRAJ22 and natural killer (NK) T-cells in the oral mucosa and lymph nodes. Our model demonstrated that Cr-specific T-cells and potent NKT-cell activation may be involved in the immune responses of Cr-induced intraoral metal contact allergy.

Palladium (Pd) is a component of several alloy types that are widely used in our environment, including several dental alloy types that cause adverse reactions such as hypersensitivity in the oral mucosa. However, the pathological mechanism of intraoral Pd allergies remains unclear because its animal model in the oral mucosa has not been established. In this study, we established a novel murine model of Pd–induced allergies in the oral mucosa, and explored the immune response of cytokine profiles and T cell diversity in terms of the T cell receptor. The Pd–induced allergy mouse was generated by two sensitizations with PdCl₂, plus a lipopolysaccharide solution into the postauricular skin followed by a single Pd challenge of the buccal mucosa. Significant swelling and pathological features were histologically evident at five days after the challenge, and CD4–positive T cells producing high levels of T helper 2 type cytokines had accumulated in the allergic oral mucosa. Characterization of the T cell receptor repertoire in Palladium allergic mice indicated that Pd–specific T cell populations were limited in V and J genes but were diverse at the clonal level. Our model demonstrated that a Pd–specific T cell population with Th2 type response tendencies may be involved in the Pd–induced intraoral metal contact allergy.